The NINDS Stroke Program Review Group identified proteolytic pathology in the neurovascular unit as a high priority research area. In this project, we will investigate the mechanisms of matrix metalloproteinase-9 (MMP-9) mediated cell death in cells of the neurovascular unit (cerebral endothelial cells, astrocytes, neurons). We hypothesize that after ischemia, MMP-9 degrades neurovascular matrix and disrupts cell-matrix interactions, thus triggering anoikis-like cell death in endothelium, astrocytes, and neurons. Hence, targeting signaling pathways that mediate MMP-9 dysregulation or initiating a rescue of homeostatic matrix signaling may be viable therapeutic approaches for stroke. To test this hypothesis, we will pursue 3 Specific Aims. In Aim 1, we examine mechanisms of MMP-9 regulation in cell cultures of cerebral endothelial cells, astrocytes, and neurons. Responses to hypoxia/reoxygenation and oxygen-glucose deprivation will be assessed. The roles of three major MAP kinases (ERK, p38, JNK) as signaling mediators will be examined. Endothelial activation via adhesion molecules will be explored as a molecular amplifier. In Aim 2, we test the idea that MMP-9 upregulation disrupts matrix integrin signals and triggers anoikis like death in endothelial cells, astrocytes, and neurons. We will examine the roles of integrin linked kinase (ILK) and Akt cell survival signaling, and try to prevent caspase-mediated cell death by integrin rescue. Pharmacologic inhibitors, RNA interference, and mutant cells lacking MMP-9 or overexpressing the endogenous inhibitor TIMP1 will be used. In Aim 3, we test the hypothesis that targeting MAP kinase pathways in mouse models of focal cerebral ischemia are protective by down regulating ischemic MMP-9 responses. U0126, SB203580, and SP600125 will be used to inhibit ERK, p38 and JNK MAP kinase pathways respectively. To complement these drugs, we will also use molecular approaches involving a JIP1 cDNA to block JNK and a Ras-GRF knockout against ERK. To assess a specific role for MMP-9, experiments will be performed in MMP-9 knockouts, transgenic mice that overexpress the endogenous MMP-9 inhibitor TIMP1, and transgenic mice that express LacZ driven by the MMP-9 promoter. Our lab and others have established MMP-9 as a key extracellular protease that modifies neurovascular homeostasis. These proposed studies should help reveal the mechanisms of MMP-9 mediated cell death in stroke.